PROJECT SUMMARY/ABSTRACT Enzymes are widely used as biocatalysts during the production of pharmaceuticals and other industrially important small molecules. In addition to operating under mild reaction conditions, enzymes are highly selective, generating enantioenriched products and minimizing side-reactions. Directed evolution is a powerful strategy for biocatalyst development, and recently it has been applied towards the engineering of hemoproteins to mediate non-natural reactions involving carbenes. However, most of these enzymes form carbenes only from ?-diazoester substrates, limiting their synthetic potential. The focus of this proposal is to expand on the synthetic and mechanistic scope of hemoproteins by evolving them to catalyze additional non-natural reactions and to characterize the determinants of chemoselectivity of these enzymes. The specific aims are (1) to evolve hemoprotein variants capable of catalyzing stereoselective Wolff rearrangements of ?-diazocarbonyl substrates, (2) to synthesize a panel of ?-diazocarbonyl compounds and use them to screen for hemoproteins useful for the production of therapeutically relevant small molecules, and (3) to biochemically characterize the factors underlying the chemo-, regio-, and enantioselectivity of several different carbene transferases. The Wolff rearrangement is a synthetically powerful tool, and biocatalysts capable of mediating asymmetric Wolff rearrangements would be valuable for constructing small molecules important to human health. Screening of known hemoprotein carbene transferases and directed evolution of new variants will be used to develop biocatalysts mediating this rearrangement. Syntheses of additional substrates will be performed to accelerate screening and to tune biocatalysts to act on industrially important small molecules using adaptive evolution. Furthermore, hemoprotein carbene transferases catalyze a wide range of reactions, and biochemical characterization and molecular dynamics simulations of these highly chemoselective enzymes will reveal the underlying features that distinguish these proteins. Identifying the molecular mechanisms influencing selectivity for these enzymes will empower future engineering efforts for developing biocatalysts catalyzing additional reactions of biological and biomedical importance.